Design of bio-based supramolecular structures through self-assembly of α-lactalbumin and lysozyme

Abstract

Bovine a-lactalbumin (a-La) and lysozyme (Lys), two globular proteins with highly homologous tertiary structures and opposite isoelectric points, were used to produce bio-based supramolecular structures under various pH values (3, 7 and 11), temperatures (25, 50 and 75 C) and times (15, 25 and 35 min) of heating. Isothermal titration calorimetry experiments showed protein interactions and demonstrated that structures were obtained from the mixture of a-La/Lys in molar ratio of 0.546. Structures were characterized in terms of morphology by transmission electron microscopy (TEM) and dynamic light scattering (DLS), conformational structure by circular dichroism and intrinsic fluorescence spectroscopy and stability by DLS. Results have shown that protein conformational structure and intermolecular interactions are controlled by the physicochemical conditions applied. The increase of heating temperature led to a significant decrease in size and polydispersity (PDI) of a-LaeLys supramolecular structures, while the increase of heating time, particularly at temperatures above 50 C, promoted a significant increase in size and PDI. At pH 7 supramolecular structures were obtained at microscale e confirmed by optical microscopy e displaying also a high PDI (i.e. > 0.4). The minimum size and PDI (61 ± 2.3 nm and 0.14 ± 0.03, respectively) were produced at pH 11 for a heating treatment of 75 C for 15 min, thus suggesting that these conditions could be considered as critical for supramolecular structure formation. Its size and morphology were confirmed by TEM showing a well-defined spherical form. Structures at these conditions showed to be stable at least for 30 or 90 days, when stored at 25 or 4 C, respectively. Hence, a-LaeLys supramolecular structures showed properties that indicate that they are a promising delivery system for food and pharmaceutical applications.

Design of β-lactoglobulin micro- and nanostructures by controlling gelation through physical variables

“…Spectra were baseline-corrected by subtracting blank spectra (i.e. 25 mmol L −1 sodium phosphate buffer) according the procedure adopted by Monteiro et al (2016) and normalized with unheated β-Lg. The maximum intrinsic fluorescence intensity was given as the average of nine successive measurements.…”

“…The samples were then negatively stained with uranyl acetate (2% w/w) (Merck, Germany) for 15 s. The grid was finally air dried at room temperature before introducing it in the electron microscope. These conditions were used based in procedures usually adopted by our research group (Monteiro et al, 2016;Bourbon et al, 2015;Pinheiro et al, 2015).…”

“…Thus, purified β-Lg was extensively characterized through a complementary set of techniques and the protein structural (conformational) changes were assessed upon thermal gelation, under combined physical and environmental conditions (various β-Lg concentrations, heating temperatures and times) at distinct pH values (3 -7), to design food-grade micro-(200 -300 nm) and nanostructures (≤ 100 nm). The small size of micro-and nanostructures can impart significant changes to physicochemical properties, among them a high surface/volume ratio, smaller pore size and increase in the solubility when compared with macroscale structures formed from the same proteins (Aklakur, Rather, & Kumar, 2016;Monteiro et al, 2016). In this regard, the effect of aforementioned physical conditions on β-Lg structures were evaluated in order to provide detailed information about the specific conditions that lead to the formation of β-Lg structures at micro-and nanoscale, which hold potential to be used as delivery systems for bioactive compounds.…”

Heteroprotein complex coacervation: A generic process

“…More recently, the complexation of these two proteins was investigated in unbuffered aqueous solution adjusted at pH values ranging from 3 to 11 [71]. Micrometric aggregates were observed at pH 7, while at pHs 3 and 11 nanometric supramolecular structures were formed.…”

“…Based on isothermal titration calorimetry experiments, another independent study presents an optimal molar ratio of around 2.0 for LYS/-LA complexation [71]. Nevertheless, these authors did not quantified the LYS/-LA molar ratio in the formed final complexes.…”